1. Field of the Invention
Immunoassays have shown themselves to be extremely versatile in allowing for methods to determine the presence of a particular substance, even when a wide variety of other materials of similar or different structure are present in the unknown sample. The immunoassays rely on the ability of an antibody to specifically detect or bind to an haptenic or antigenic organic compound, while not interacting with other compounds. The divalent nature of the antibody and/or its high molecular weight, 150,000 or greater, allow for a discrimination between a compound which is bound and a compound which is not bound to antibody. Among various immunoassays involving antibodies are radioimmunoassays, spin immunoassay, homogeneous enzyme immunoassay, available under the trademark EMIT, supplied by Syva Company, and hemeagglutination.
The enzyme immunoassay is extremely versatile in permitting spectrophotometric determinations. The immunoassay employs an enzyme to which an organic compound--a ligand--is conjugated at a position where when bound to receptio, the activity of the enzyme is substantially reduced. To the extent that the unknown sample contains the same ligand the amount of receptor available for binding to the organic compound conjugated to the enzyme is reduced. Therefore, by analyzing for enzymatic activity, a significant increase in enzymatic activity over the enzymatic activity in the absence of the unknown indicates the presence of the ligand in the unknown.
The sensitivity of the homogeneous enzyme immunoassay is based to a substantial degree on the activity of the enzyme when conjugated and the degree of inhibitability when receptor is bound to the ligand conjugated to the enzyme. It is, therefore, desirable to have an enzyme which not only has a high turnover rate initially, but retains a substantial proportion of this turnover rate after conjugation, and is strongly inhibited when receptor is bound to the ligand which is conjugated to the enzyme. Also, the enzyme should allow for strong specific binding of receptor to the conjugated ligand.
2. Description of the Prior Art
An homogeneous enzyme immunoassay system has been sold under the trademark EMIT employing haptens conjugated to lysozyme, where the enzymatic activity is determined by the reduction in turbidity as a result of lysis of bacterial walls. Numerous publications concerning the system have issued since May 7, 1971, see for example, Rubenstein, et al., Biochem. & Biophysical Res. Comm. 47, 846 (1972). U.S. Pat. No. 3,654,090 teaches a heterogeneous immunoassay employing such enzymes as peroxidase, amyloglucosidase, and .beta.-D-galactosidase with antigens.